The Main Pharmacodynamics And Pharmacological Mechanism Of Therapeutic Effects On Human Gastric Cancer By Recombinant Mutant Human Tumor Necrosis Factor For Injection

Background and aim:Recombinant Mutant Human Tumor Necrosis Factor for injection (rmh-TNF NC)is an non-glycosylated single polypeptide chain consisted of 151 amino acid residue, the molecular weight is 16598 Daltons, modified TNF-a gene on the basis of cDNA of wild type TNF-a by PCR (polymerase chain reaction). With this modification, expending the scope of curative effect and enhance anti-tumor activity, raising the efficiency of joint, obtain a novel mutant recombinant human tumor necrosis factor with higher activity and lower toxicity, is referred to as the "rmh-TNF". Rmh-TNF is the mutant TNF-αwith the similar biologic functions and the selective cytotoxicity and obviously chemosensitize effects. It is the one country category of new drugs which new entranced clinical practice, the principal indications are non-small-cell lung cancer and non-Hodgkin's lymphoma; however, the reports of experimental researches on the application on gastrointestinal tumor especial gastric cancer are rare. Currently the gastric cancer death is still the world's fourth reason and second cancer incidence, an estimated 650000 deaths and 880000 new cases every year, almost two-thirds are in developing countries. This study aims to research on the main pharmacodynamics and pharmacological mechanism of therapeutic effects on human gastric cancer of recombinant mutant human tumor necrosis factor for injection, by detecting of the proliferation, apoptosis, angiogenesis, clone forming and migration ability and by western bolt and co-immunoprecipitation, respectively.Method:Culture human gastric cancer cell BGC-823 and human umbilical vein endothelial cell HUVEC-12 treat with different concentrations rmh-TNF and TNF-αin vitro, establish the control group. Detect the proliferation by MMT assay, test apoptosis by Annexin V-FITC/PI and TUNEL assay. Flow cytometric analysis of the cell cycle and apoptosis ratio. Test the angiogenesis ability of HUVEC-12 by tube formation assay test. Test clone forming and migration ability of BGC-823 by Soft-agar colony forming assay and wound-healing assay, respectively. Establish BALB/c-nude mice transplanted subcutaneously human gastric cancer model, divided into rmh-TNF, TNF-α, positive control(5-Fu) and negative control groups for therapeutic experiment, draw the growth curve of implanted tumor, count tumor weight inhibitory ratio, observe these effects on the sub-skin implanted tumor formation, the survival time and the life-prolong rate, the general condition and anatomical changes, observe the pathologicalhistology of implanted tumor by HE staining, count the microvessel density (MVD)in implanted tumor marked with CD34 by immunohistochemical staining. Investigate the expression levels of Procaspase-3, Bcl-2 and Bax in BGC-823. Detect whether there is interaction between rmh-TNF and DR5 by co-immunoprecipition.Result:MTT assay results shows that rmh-TNF can inhibit human BGC-823 and HUVEC-12 cells proliferation, presenting well dose-response and time-effect relationship, its IC50 value is half and three-fourth of TNF-α, showing a significant difference(P< 0.05).Annexin V-FITC/PI and TUNEL assay results show the dose-dependent ability of inducing apoptosis of rmh-TNF. Both types of cells treated by rmh-TNF and TNF-a cell cycle analysis emerge promoted proportion of S phase and reduced proportion of G0/G1 and G2/M phase, apoptosis ratio arising, these effects are dose-dependent.Tube formation assay shows the inhibitory effect of rmh-TNF on angiogenesis, soft-agar colony forming assay and wound-healing assay demonstrate rmh-TNF has the inhibitory effects on BGC-823 clone forming and migration ability, respectively. The nude-mice sub-skin model transplanted tumor is established successful, the growth curve of implanted tumor shows that the growth of tumor in rmh-TNF group has been inhibitory obviously, tumor weight inhibitory ratio in rmh-TNF group is 83.13%.The life-prolong rate of rmh-TNF, TNF-αand positive control group are 85.18%,62.34%, and 59.88%, respectively, the general condition of nude-mice bearing tumor in negative control group is obviously poor than treatment group and positive control group, there is no disease of organs and systems beside implanted tumor in any group of nude-mice by anatomy. There are obvious apoptosis and necrosis in treatment group and positive control group, with decreased pleomorphic cell, and lower MVD marked with CD34 by immunohistochemistry, especially the rmh-TNF group, showing a significant difference(P< 0.05). Significant higher expressions of Procaspse-3 and Bax, lower expression of Bcl-2 in rmh-TNF group compared to other group, the effects are dose-depended. Confirm there is interaction between rmh-TNF and DR5 by co-immunoprecipition. Conclusion:1. Rmh-TNF can inhibit proliferation and induce apoptosis on human gastric cancer BGC-823 and human umbilical vein endothelial cells HUVEC-12 in vitro, more than TNF-α, showing a significant difference(P＜0.05);2. Rmh-TNF can inhibit the angiogenesis ability of HUVEC-12, clone forming ability of BGC-823, more than TNF-α, showing a significant difference(P＜0.05), and can prevent BGC-823 to migrate; 3. Human gastric cancer cell BGC-823 has satisfactory characteristic with tumor formation in BALB/c nude-mice; 4. Rmh-TNF has the therapeutic effects in the nude-mice sub-skin model transplanted tumor, can inhibit the growth of implanted tumor, prolong the survival time, more than TNF-α, showing a significant difference (P＜0.05); 5. Rmh-TNF can induce apoptosis and necrosis in human gastric cancer BGC-823 BALB/c nude-mice transplanted tumor, with anti-angiogenesis effects, more than TNF-α, showing a significant difference (P＜0.05); 6.Rmh-TNF may induce apoptosis by up-regulating expression of Procaspase-3 and Bax down-regulating expression of Bcl-2, and prompting the Bax/Bcl-2 value; 7. Rmh-TNF may conduct its biological effects through combination with DR5.